The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase.

The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClpP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM. ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.     

lon gene product of Escherichia coli is a heat-shock protein

The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli.     

The dnaK protein modulates the heat-shock response of Escherichia coli

E. coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene. Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli. Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C. Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins. Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation. In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature. We conclude that the dnaK protein is an inhibitor of the heat-shock response in E. coli.     

Isolation and physical mapping of temperature-sensitive mutants defective in heat-shock induction of proteins in Escherichia coli

Summary Mutants of Escherichia coli K12 that are partially or totally defective in induction of major heat-shock proteins and cannot grow at high temperature (42° C) were isolated by localized mutagenesis. These mutants carry a single mutation in the gene htpR (formerly hin) located at min 76 on the E. coli genetic map. Some mutants exhibit delayed (partial) induction of heat-shock proteins or require a higher temperature for induction than the wild type, whereas others are not induced under any of these conditions. The maximum temperature that allows growth varies among different mutants and is correlated with the residual induction capacity. Temperature-resistant revertants obtained from each mutant are fully or partially recovered in heat-shock induction. These results indicate that the inability of htpR mutants to grow at high temperature is due to the defect in heat-shock induction. In addition, a couple of mutants was found that produce significantly higher amounts of heat-shock proteins even at 30° C.The htpR gene has been cloned into plasmid pBR322 using the above mutants, and was localized to a DNA segment of 1.6 kilobase pairs. The mutants harboring certain palsmids that carry a part of htpR produce temperature-resistant recombinants at high frequency. This permits further localization of mutations within the htpR gene. Analysis of proteins encoded by each of the recombinant plasmids including the one carrying a previously isolated amber mutation (htpR165) led to the identification of a protein with an apparent molecular weight of about 36,000 daltons as the htpR gene product.     

The chaperonin GroEL and other heat-shock proteins, besides DnaK, participate in ribosome biogenesis in Escherichia coli

It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo. Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature. GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly. Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C.     

Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.

The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.     

Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down.

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down.     

The synthesis of export-defective proteins can interfere with normal protein export in Escherichia coli

We have analyzed the kinetics of maturation for certain bacterial envelope proteins in Escherichia coli strains that are also concomitantly producing an export-defective protein. Our data indicate that proteins with defective signal peptides, rendered nonfunctional by either point mutation or deletion, interfere with the normal export of other envelope proteins. Expression of interference requires that the interfering protein: (i) exhibit a major export defect; (ii) be synthesized at a high rate; and (iii) be actively synthesized at the time interference is being measured. The latter data suggest that interference is a cotranslational process. Intragenic or extragenic suppression of the export defect exhibited by the interfering protein relieves interference in a manner that is directly related to strength of suppression. These and additional data suggest that interference occurs at a very early step in the secretory process. We interpret these results to indicate that proteins with defective signal peptides are still recognized as proteins destined for secretion and are, therefore, at least transiently incorporated into the cell's secretory pathway. The incorporation of an export-defective protein into the secretory pathway disrupts the normal protein traffic from the cytoplasm to the various extracellular compartments.     

The relationship of heat-shock proteins, thermotolerance, and protein synthesis

The relationship of heat-induced inhibition of protein synthesis (HIIPS) and thermotolerance, the transient ability to survive otherwise lethal heat treatments, was studied in HA-1 Chinese hamster fibroblasts exposed to various treatments. A mild heatshock or exposure to sodium arsenite induced a refractoriness to HIIPS, while exposure to the amino acid analog of proline, azetidine, did not. The development and decay of refractoriness to HIIPS after exposure to heat or sodium arsenite paralleled in the increase and decrease of the rate of synthesis of the heat-shock proteins (HSP), and was associated with neither the persistence of elevated levels of HSP nor the persistence of the thermotolerant state. Refractoriness to HIIPS was not associated with the elevated synthesis of HSP in the presence of amino acid analogs regardless of the mode of induction, indicating a requirement for functional HSP for the effect. The refractoriness to HIIPS was also found in heat-resistant variants of HA-1 cells that express elevated levels of hsp 70, implicating a role for this protein in this process. Our observation establish an unique biological effect associated with the period of elevated synthesis of the HSP, especially the hsp 70.     

Chemical synthesis and in vivo hyperexpression of a modular gene coding for Escherichia coli translational initiation factor IF1

Summary An artificial gene encoding the Escherichia coli translational initiation factor IF1 was synthesized based on the primary structure (71 amino acid residues) of the protein. Codons for individual amino acids were selected on the basis of the preferred codon usage found in the structural genes for the initiation factor IF2 of E. coli and Bacillus stearothermophilus, both of which can be expressed at high levels in E. coli cells. We gave the IF1 gene a modular structure by introducing specific restriction enzyme sites into the sequence, resulting in units of three to ten codons. This was conceived to facilitate site-directed mutagenesis of the gene and thus to obtain IF1 with specific amino acid alterations at desired positions. The IF1 gene was assembled by shot-gun ligation of 9 synthetic oligodeoxyri-bonucleotides ranging in size from 31 to 65 nucleotides and cloned into an expression vector to place the gene under the control of an inducible promoter. Upon induction, E. coli cells harbouring the artificial gene were found to produce large amounts (60 mg/100 g cells) of a protein indistinguishable from natural IF1 in both chemecal and biological properties.